Lipoxygenases are a family of non-heme iron dioxygenases that catalyze the stereo- and regio-specific formation of fatty acid hydroperoxides from polyunsaturated fatty acids (35, 36). In addition to 5-LOX, which catalyzes the peroxidation of arachidonic acid (AA) at the C5 position, mammalian lipoxygenases that form the 12-, 15- and 8-hydroperoxide products of AA oxygenation (hydroperoxyeicosatetraenoic acid, HPETE) have also been described. Their products are converted to other oxylipins with diverse roles in biology (see (35)). Lipoxygenases are also widely distributed throughout the plant kingdom (35), but the substrates for the plant enzymes are generally the 18 carbon linoleic and linolenic acids rather than the 20 carbon AA recognized by animal enzymes. The first step of a LOX-catalyzed reaction is hydrogen abstraction at the central carbon of a pentadiene moiety by the activated Fe3+ (37) to produce a free radical intermediate which is oxygenated two carbons removed from the position of hydrogen abstraction. Active site control of regio-specificity is determined by which pentadiene of the substrate is positioned for attack, and whether O2 has access to carbon C−2 or C+2. Animal lipoxygenases are named according to their product specificity; while AA is the substrate for all of them, the position and stereochemistry of the hydroperoxy group introduced is specific for a given isoform. 5-LOX catalyzes both the dioxygenation of an unsaturated fatty acid to its hydroperoxy derivative, the reaction common to all lipoxygenases, and the subsequent transformation of the 5-hydroperoxyeicosatetraenoic acid (5-HPETE) to leukotriene A4, in which one of the oxygen atoms of the hydroperoxide ends up in an epoxide. The first reaction requires abstraction of hydrogen at C7, while the second reaction requires abstraction of the hydrogen at C10.
In the human body, 5-LOX is used to produce pro-inflammatory leukotrienes, which are potent lipid mediators of the inflammatory response. As stated above, 5-LOX catalyzes a two step transformation of (1) arachidonic acid (AA) at the 5-position to yield 5-hydroperoxyeicosatetraenoic acid (5-HPETE), and then (2) 5-HPETE to leukotriene A4. Leukotrienes are potent lipid mediators of the inflammatory response, including the response involved in asthma. Over the last 25+ years, substantial progress has been made in understanding how leukotrienes exert their effects, and a broader appreciation for the numerous biological processes that leukotrienes mediate has resulted. For example, 5-LOX has been linked to development of heart disease, stroke and atherosclerosis.
Leukotrienes (LT) and lipoxins are potent mediators of the inflammatory response derived from arachidonic acid (AA). When leukocytes are activated, arachidonic acid is released from the nuclear membrane by the action of cytosolic phospholipase A2 and binds five-lipoxygenase-activating protein (FLAP). The increased Ca2+ concentration of the activated cells simultaneously promotes translocation of 5-LOX to the nuclear membrane where it acquires its substrate from FLAP (1, 2). Arachidonic acid (AA) is converted to leukotriene (LTA4) in a two-step reaction which produces the 5S-isomer of hydroperoxyeicosatetraenoic acid (5S-HPETE) as an intermediate (3, 4).
Work with the plant enzymes has afforded tremendous insight into the mechanism of hydrogen abstraction by the active site iron, but the basis for regio-specificity of the animal enzymes is still unclear. The 1.85 Å resolution structure of an 8R-lipoxygenase has been described (7, 10). In addition, there is a structure available for the 15S-enzyme from rabbit reticulocytes (11, 12).
Regulatory mechanisms that provide the transient activities associated with temporal control of cellular events include targeted degradation, phosphorylation, and allosteric control of enzyme activities. Auto-inactivation that is a consequence of intrinsic protein (in)stability can also have a role in temporal control of protein function. For example, the relative instability of the tumor suppressor protein p53, relative to its orthologs such as p73, has been proposed to a have a functional role (9).
Auto-inactivation has been proposed to play an important regulatory role in mammalian 5-lipoxygenase (5). As mentioned above, in mammals LOX products are the precursors of potent lipid mediators of the inflammatory response; thus an overproduction of the signaling compounds is detrimental to the organism. Enzyme lability, whether a consequence of turnover or non-turnover-based inactivation, serves as an auto-shutoff valve, an innate “programmed obsolescence.” In contrast, the 8R-LOX from Plexaura homomalla is remarkably stable, perhaps an indication that constitutive production of LOX products is beneficial to the soft coral (7). Human 5-LOX and P. homomalla 8R-LOX represent two extremes of lipoxygenases. While these enzymes share 40% sequence identity, and consequently a protein fold, they differ significantly in their inherent stabilities, with 5-LOX a notoriously unstable enzyme, and the 8R-enzyme remarkably robust. Yet the enzymes recognize the same substrate, utilize the same catalytic machinery, and perform a common chemical transformation. Furthermore, both are targeted to the membrane in a Ca2+-dependent fashion. The Ca2+-binding amino acids, as well as putative membrane insertion loops, are shared by these two lipoxygenases, but absent in all other lipoxygenase isoforms.
Purified, human 5-LOX is unstable, having a half-life as short as 10-hours at 4° C. (8). In addition, its low solubility and “stickiness” frustrates handling of the enzyme. Native 5-LOX variants have a tendency to “clump” when placed in aqueous solutions, which leads to wasteful residues being left on containers, such as glass beakers. A soluble and/or stable form of human 5-LOX is highly desirable.